The major objective of our research is to delineate the molecular mechanism(s) of tissue- and stage-specific, multi-hormone regulated gene expression using the beta-casein gene as a tool. beta-Casein is the major milk protein produced by the lactating mammary gland. The transcription of casein gene requires the synergistic actions of insulin, prolactin, glucocorticoid, and EGF. Transfection experiments using reporter plasmids containing various 5'-flanking region of the mouse beta-casein gene and primary mouse mammary epithelial cell cultures indicated that the 252 bp long promoter region is sufficient for hormonal induction. This region contains three highly conserved sequences, designated as blocks A, B, and C, in the promoters of many casein gnes. Previous studies showed that block B and probably Block A serve as an acting site of a prolactin-dependent transcription factor, mammary gland-specific factor (MGF). These findings suggest that the highly conserved sites of the casein gene promoter play a major role in hormonal induction of casein gene transcription. Since the role of block C in regulating casein gene transcription has not been elucidated, we have carried a series of experiments to assess its function. Transfection experiments using various reporter constructs bearing mutations within block C indicated that block C, like the other two conserved regions, is crucial for the beta-casein gene transcription induced by lactogenic hormones. Our studies also revealed the presence of nuclear protein complexes in the lactating mouse mammary gland that specifically bind to both double-stranded (DS) and single-stranded (SS) DNA within block C. The maximal increase in the binding activity of DS and SS in the mammary gland occurred during pregnancy and during lactation, respectively, In organ culture, the DS activity is increased by EGF or prolactin in combination with insulin, whereas the SS activity is enhanced by insulin, prolactin and glucocorticoid. These results suggest that the hormonal induction of beta-casein gene expression involves stimulation of DS and SS binding activites, which in turn, enhance the gene transcription via block C.